Nerve specificity combined detection composition for cognitive disorder assessment

Abstract

The invention relates to the technical field of medical preparations, and discloses a nerve specificity combined detection composition for cognitive disorder assessment, which comprises a hydrophobic fluorescent probe as a dispersed phase and an active oxygen responsive amphiphilic block copolymer as a continuous phase medium, the copolymer is self-assembled in an aqueous medium to form a micelle with a hydrophobic core, and the probe is entrapped in the core; the mass ratio of the probe to the copolymer and the molecular weight of a hydrophobic chain segment in the copolymer are limited, so that the probe forms a thermodynamically metastable H-aggregate in the inner core, the metastable structure is utilized, the composition keeps low background fluorescence in the transportation process, the probe is disassembled and released when the composition responds to a pathological environment, and the detection sensitivity is improved. And high-sensitivity assessment of the cognitive impairment risk is realized.

Description

Technical Field

[0001] This invention relates to a neurospecific combined detection composition for the assessment of cognitive impairment, belonging to the field of medical preparations technology. Background Technology

[0002] Current early screening for cognitive impairment utilizes fluorescent probes to target and detect retinal beta-amyloid protein deposition. To improve the bioavailability of hydrophobic probes and reduce non-specific toxicity, existing technologies employ amphiphilic block copolymer micelle carriers, utilizing environmental response mechanisms to release probes that label pathological targets.

[0003] Constructing micellar physical carriers cannot solve the problem of matching probe release kinetics with the time history of transient pathological signals. Existing technologies for regulating the phase state of the core material follow the steady-state concept of drug delivery. For example, Chinese invention patent CN102670525A discloses the use and preparation of ursolic acid nanoparticles for tumor treatment. Although it uses polycaprolactone-polyethylene glycol block copolymer, it utilizes the tightly wrapped polymer chain structure to build thermodynamically stable crystalline microregions to achieve long-term sustained drug release and stable in vivo circulation. This technology, which reduces the release rate and pursues high lattice energy stability, meets the need for sustained blood drug concentration in tumor treatment. However, when applied to the field of neural signal detection, it presents a kinetic barrier. Facing the millisecond to second-level transient oxidative stress fluctuations of retinal neurons, the formation of dense microcrystalline probes requires overcoming the high lattice energy barrier to dissociate into monomers. Formulation application requires balancing fluorescence quenching efficiency and release kinetics. Suppressing background fluorescence during transport generally increases the probe loading density in the micelle core, causing probe molecules to pack tightly into H aggregates. After non-radiative quenching, the high concentration of hydrophobic planar molecules in the confined space has a spontaneous crystallization tendency, causing probe molecules to form dense microcrystals or nanocrystals in the micelle core. The microcrystal structure introduces a release kinetic barrier. When the micelle carrier responds to the structural disintegration of the pathological microenvironment, the core probe microcrystals need to overcome the lattice energy to dissociate into monomer molecules. The dissociation process takes several minutes. Early oxidative stress bursts or transient neurochemical fluctuations in retinal neurons occur in milliseconds to seconds. The mismatch between the carrier response and probe release time makes it difficult for the formulation to capture transient pathological signals. Reducing the loading amount avoids crystallization and prevents the formation of H aggregates for quenching, thus preventing background fluorescence leakage during transport.

[0004] Therefore, how to construct an ophthalmic detection composition that maintains fluorescence quenching while eliminating crystal lattice energy binding, and achieves millisecond-level response to pathological microenvironment signals, has become the technical problem to be solved by this invention. Summary of the Invention

[0005] To address the problems raised in the background art, the technical solution of the present invention is as follows: a neurospecific combined detection composition for cognitive impairment assessment, the composition being an ophthalmic preparation comprising a dispersed phase and a continuous phase medium; The dispersed phase is a hydrophobic fluorescent probe, and the continuous phase medium is a reactive oxygen species-responsive amphiphilic block copolymer; The reactive oxygen species responsive amphiphilic block copolymer is methoxy polyethylene glycol-thioketal-polycaprolactone, which assembles in an aqueous medium to form micelles with a hydrophobic core, and the hydrophobic fluorescent probe is encapsulated in the hydrophobic core. Among them, the number average molecular weight of the polycaprolactone segment in methoxy polyethylene glycol-thioketone-polycaprolactone is limited to 1800 Da to 2200 Da, so as to form a semi-crystalline micro-region that physically confines the hydrophobic fluorescent probe in the hydrophobic core. The mass ratio of the hydrophobic fluorescent probe to the reactive oxygen species-responsive amphiphilic block copolymer is limited to 8:100 to 12:100; Based on the physical confinement effect of mass ratio and polycaprolactone segments, the hydrophobic fluorescent probe forms thermodynamically metastable H-aggregates in the hydrophobic core. The thermodynamically metastable H-aggregates do not have sharp diffraction peaks corresponding to the single crystal of the hydrophobic fluorescent probe in the X-ray powder diffraction pattern, but show diffuse peaks of the polymer matrix. Moreover, the maximum absorption wavelength in the UV-Vis absorption spectrum is blue-shifted by 30 nm to 50 nm relative to the monomer solution of the hydrophobic fluorescent probe. When the thioketal bond breaks in response to pathological concentrations of reactive oxygen species, the hydrophobic fluorescent probe is released along with the micelle disintegration and binds to the target amyloid protein.

[0006] Preferably, the hydrophobic fluorescent probe is selected from one of the modified curcumin derivatives or boron fluoride dipyrrole derivatives with molecular rotor characteristics, whose molecular structure contains at least two rigid conjugated planes capable of forming π-π stacking; the number average molecular weight of the methoxy polyethylene glycol segment in methoxy polyethylene glycol-thioketal-polycaprolactone is 1900 Da to 2100 Da; the fluorescence quantum yield of the composition in the thermodynamically metastable H-aggregate state is less than 1%, and the fluorescence recovery response time after the thioketal bond breaks is less than 100 ms; the particle size distribution coefficient of the composition is less than 0.2.

[0007] Preferably, the composition is prepared by an antisolvent precipitation process, the process comprising: dissolving a hydrophobic fluorescent probe and a reactive oxygen species-responsive amphiphilic block copolymer in tetrahydrofuran to form a homogeneous solution, and injecting the homogeneous solution into deionized water at a flow rate of 10.0 mL / min to 12.0 mL / min while stirring at a speed of 1000 rpm to 1200 rpm; and fixing the hydrophobic fluorescent probe in a thermodynamically metastable H-aggregate state by matching the flow rate with the precipitation rate of the polycaprolactone segments.

[0008] Preferably, the background fluorescence suppression ability of the composition is determined by the fluorescence quenching efficiency. Characterization, fluorescence quenching efficiency The following relationship must be satisfied: ,in, The fluorescence emission intensity of the composition at the excitation wavelength is measured when the composition is in an intact micelle state. The fluorescence emission intensity is the value of the composition after the thioketal bond is completely broken and the hydrophobic fluorescent probe is completely released into the octanol medium.

[0009] Preferably, the volume ratio of tetrahydrofuran to deionized water is 1:5 to 1:10; the process also includes open-air stirring of the mixed solution at an ambient temperature of 30 degrees Celsius to remove tetrahydrofuran, and dialysis purification of the micelle solution using a dialysis unit with a molecular weight cutoff of 3500 Da; the thermodynamically metastable H-aggregates are amorphous structures formed based on solvent displacement and competition for crystallization of polycaprolactone segments.

[0010] Preferably, the composition further includes a pharmaceutically acceptable isotonic buffer solution with a pH of 7.0 to 7.4; the composition exhibits a fluorescence leakage rate of less than 2% within 30 days when stored at 4°C in the dark; and the hydrophobic fluorescent probe reacts with... When amyloid plaques bind, their fluorescence emission intensity increases by at least 100 times relative to the thermodynamically metastable H-aggregate state before release, achieving a dual specific response to oxidative stress and amyloid deposition.

[0011] Preferably, the critical micelle concentration of the reactive oxygen species-responsive amphiphilic block copolymer is [value missing]. mg / mL to mg / mL; the thioketal bond is located at the junction of the methoxy polyethylene glycol segment and the polycaprolactone segment, and is used to chemically break in an environment where the concentration of hydrogen peroxide or hydroxyl radicals exceeds 100 μmol / L, thereby leading to micelle disassembly.

[0012] Preferably, the emission wavelength of the hydrophobic fluorescent probe is located in the region of 650 nm to 800 nm, and its Stokes shift is greater than 80 nm; the intermolecular distance of the hydrophobic fluorescent probe in the thermodynamically metastable H-aggregate state is restricted by polymer chain segments to between 0.3 nm and 0.4 nm, which is less than the lattice spacing required to form a stable crystal but sufficient to induce intermolecular interactions to achieve fluorescence quenching.

[0013] Preferably, the composition is used to prepare a fundus diagnostic reagent for assessing the risk of Alzheimer's disease or mild cognitive impairment. The reagent is delivered to the retina via eye drops to avoid nonspecific fluorescence interference from the lens and vitreous body.

[0014] Preferably, the composition is a colloidal solution with a Zeta potential of -10mV to -20mV; the residual organic solvent content in the composition is less than 50 parts per million; the thermodynamically metastable H-aggregates still maintain the amorphous characteristics without sharp crystal diffraction peaks in the X-ray powder diffraction pattern after three freeze-thaw cycles, indicating that the polycaprolactone chain segment has reversible and stable crystallization inhibition effect on the hydrophobic fluorescent probe.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. In neurospecific joint detection, by limiting the mass ratio of fluorescent probes to amphiphilic block copolymers and the molecular weight range of hydrophobic polymer segments, a metastable phase of frustrated H-aggregates is constructed in the hydrophobic core of micelles. Utilizing the spatial intercalation and confinement effects of polymer segments, the thermodynamic tendency of high-concentration probe molecules to transform into long-range ordered crystals is suppressed, maintaining the dense π-π stacking state between probe molecules to sustain fluorescence quenching. The specific microscopic physical structure enables the composition to maintain optical silence during transport, eliminating the need to overcome the high lattice energy barrier associated with probe microcrystallization in traditional high-load formulations. When the micelle shell breaks in response to the pathological environment, the core probe directly skips the slow crystal dissolution step, achieving instant phase separation and release, thus solving the signal response delay problem caused by crystallization in existing ophthalmic hydrophobic fluorescent formulations.

[0016] 2. The composition utilizes the reactive oxygen species-responsive chemical bond breaking mechanism and the viscosity-sensitive luminescence properties of the molecular rotor probe to construct a logic and gating system at the molecular level targeting the pathological characteristics of cognitive impairment. When the target area has oxidative stress environment indicating neurotoxicity and amyloid plaques indicating structural lesions, it triggers a complete signal transduction process from carrier disassembly to probe binding and luminescence. Based on the dual locking mechanism of the chemical properties of the substance, the detection signal is limited to the lesion area with active pathological toxicity, shielding the false positive interference caused by benign aging tissue with only protein deposition but no oxidative stress activation, thereby improving the accuracy of the composition in assessing the risk of pathological cognitive impairment.

[0017] 3. The restricted antisolvent kinetic capture process enables the core probe molecules of the composition to store high Gibbs free energy, establishing a release kinetic characteristic driven primarily by entropy increase. When the hydrophilic shell of the micelle is cleaved by reactive oxygen species at the lesion site, the probe molecules, which are in a high-energy metastable state, are not constrained by the stability of crystal growth and migrate explosively to the hydrophobic microenvironment of the adjacent amyloid plaque on a millisecond timescale. This kinetic characteristic, based on the composition's extremely high temporal resolution, captures the early, weak, and short-term transient oxidative stress fluctuations of retinal neurons, overcoming the technical problem of existing thermodynamically stable formulations being unable to detect early dynamic pathological features due to their excessively large response time constant. Attached Figure Description

[0018] Figure 1This is a flowchart illustrating the physical confinement preparation process of the composition of the present invention and the mechanism of reactive oxygen species-responsive fluorescence enhancement. Figure 2 This is a comparison chart of fluorescence quenching efficiency and response signal enhancement factor performance for different parameter sample groups of the present invention; Figure 3 This diagram illustrates the microscopic disassembly and specific fluorescence recovery mechanism of the composition of the present invention from the transport state to the release state. Detailed Implementation

[0019] This specific embodiment is intended to provide a detailed description of the technical solution of the present invention, but should not be construed as limiting the scope of protection of the present invention. Any obvious changes or substitutions made by those skilled in the art based on the teachings of the present invention shall fall within the scope of protection of the present invention.

[0020] This invention provides a neurospecific combined detection composition for cognitive impairment assessment, comprising an amphiphilic block copolymer responsive to reactive oxygen species (ROS). This copolymer self-assembles into nanomicelles in an aqueous medium. The dispersed phase consists of hydrophobic fluorescent probes physically encapsulated within the hydrophobic core of the micelles. By limiting the component mass ratio and the length of the hydrophobic segments of the copolymer, combined with a restricted antisolvent precipitation process, the composition maintains the core probe molecules in a thermodynamically metastable frustrated H-aggregate state. In this state, the close packing of probe molecules leads to fluorescence quenching. When the micelle shell undergoes chemical cleavage in response to ROS in the environment, the metastable probe molecules undergo phase separation and release driven by entropy increase, binding to amyloid plaques and restoring fluorescence radiation transitions. To ensure the formation of the frustrated H-aggregate core, the core components constituting the micelles must meet specific physicochemical parameter limitations. The carrier material of the micelles is a methoxy polyethylene glycol-thioketal-polycaprolactone block copolymer. In this copolymer, the polycaprolactone segments, as the hydrophobic ends, have a number-average molecular weight of... Limited to a molecular weight range of 1800 Da to 2200 Da, the length of the polycaprolactone segments within this range spatially matches the aggregate size of the probe molecules, providing hydrophobic microdomains and restricting long-range ordered crystallization of the probe molecules. The methoxylated polyethylene glycol segments at the hydrophilic ends have a number-average molecular weight of 1900 Da to 2100 Da, used to maintain the colloidal stability of the micelles. The thioketal bonds connecting the two blocks are chemically sensitive to hydrogen peroxide or hydroxyl radicals, breaking upon contact. The hydrophobic fluorescent probe is CRANAD-2 or its structural analogues, whose molecular structure contains at least two molecules capable of forming... The probe, consisting of stacked rigid conjugated planes and possessing molecular rotor properties, exhibits photoluminescence in a monomeric dispersed state, fluorescence quenching in a tightly packed state, and fluorescence emission recovery upon embedding in the high-viscosity hydrophobic environment of amyloid plaques.

[0021] The composition was prepared using a confined antisolvent kinetics capture process, comprising the following steps: At 25°C, 100.0 mg of the copolymer and 8.0 mg to 12.0 mg of the hydrophobic fluorescent probe were dissolved in 2.0 mL of tetrahydrofuran. This 8:100 to 12:100 mass ratio is the basis for the formation of frustrated H-aggregates. The mixture was sonicated at 40 kHz for 5 minutes to obtain a homogeneous solution. A reaction system including magnetic stirring was established. 10.0 mL of deionized water was placed in a container, and the temperature was maintained at 25°C. The stirring speed was set to 1000 rpm to 1200 rpm. The tetrahydrofuran mixture was pumped into the deionized water at a constant flow rate of 10.0 mL / min to 12.0 mL / min using a syringe pump. This injection flow rate matched the hydrophobic collapse rate of the polycaprolactone segments, fixing the probe molecules in an amorphous frustrated H-aggregate state. The first step of the process was to establish a steady-state flow field model for the solvent displacement region and set the stirring Reynolds number according to the Navier-Stokes equation. Maintain at to The entire turbulent range, in which For fluid density, For stirring speed, For the diameter of the agitator, For fluid dynamic viscosity, ensure micro-mixing timescale Less than the critical nucleation time of polycaprolactone segments This causes hydrophobic collapse of the chain segments before they complete their lattice arrangement; the input parameter calibration process requires real-time monitoring of the torque change rate within the reactor. ,when Exceed The system was immediately identified as a precursor to macroscopic precipitation due to a sudden change in local viscosity, and the logic control unit promptly adjusted the flow rate of the injection pump. Following the negative feedback algorithm The gain coefficient Set as During the output acceptance phase, online dynamic light scattering monitoring is introduced, and the polydispersity index (PDI) threshold is set to [value missing]. If real-time monitoring value And the decay rate of the light intensity autocorrelation function Deviation from preset baseline If the micelle nuclei in this batch are found to have undergone unrestricted crystallization, the waste liquid diversion valve will be automatically triggered, thereby achieving closed-loop control for the capture of metastable structures.

[0022] The resulting micelle suspension was stirred openly at 30°C for 6 hours to remove tetrahydrofuran. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in 2000 mL of deionized water for 24 hours, with the dialysate changed every 6 hours. Finally, an isotonic adjuster was added to adjust the osmotic pressure to 280 mOsm / kg to 320 mOsm / kg and the pH to 7.0 to 7.4. The solution was then passed through a pore size... Microporous membrane filtration; the material state of the probe molecules in the composition is defined by the following physical indicators, in the X-ray powder diffraction pattern, the scanning range for to At that time, the lyophilized powder of the composition did not show any sharp diffraction peaks corresponding to the probe single crystal, but only diffuse peaks of the polymer matrix. In the UV-Vis absorption spectrum, the maximum absorption wavelength of the aqueous solution of the composition was... A blue shift of 30 nm to 50 nm occurs relative to the probe in the monomer solution in tetrahydrofuran; during transport, the composition maintains an intact micelle morphology, the core probe is in a frustrated H-aggregate state, and the fluorescence quantum yield is less than 1%, even at contact concentrations exceeding [missing value]. In a hydrogen peroxide environment, the thioketal bonds break, leading to micelle disassembly. Probe molecules undergo phase separation and release driven by entropy increase, and bind to... -Amyloid plaques, after binding, restrict the intramolecular rotation of probe molecules due to steric hindrance, and the fluorescence emission intensity increases by at least 100 times compared to the aggregated state before release.

[0023] Example 1: When the composition of the present invention is applied to the capture of trace signals characteristic of early neurodegenerative retinal diseases, the detection system faces a contradiction between the non-specific adsorption caused by the intrinsic high lipophilicity of the fluorescent probe and the weak signal in the lesion area. Under this condition, in the retinal tissue... - Amyloid plaques are on the micrometer scale and are accompanied by transient oxidative stress fluctuations. This requires the detection agent to maintain absolute optical silence during transport through the lipid- and protein-rich vitreous humor and achieve a millisecond-level signal response upon contact with the lesion. Unlike conventional methods that reduce probe concentration to suppress background noise, this approach constructs a frustrated H-aggregate phase and utilizes intermolecular interactions under high concentration loading to achieve fluorescence self-quenching. Specifically, the number-average molecular weight of the polycaprolactone segments in the methoxy polyethylene glycol-thioketal-polycaprolactone copolymer... Limited to a concentration of 1800 Da to 2200 Da, and with a hydrophobic fluorescent probe loading ratio of 8% to 12%, the spatial topology of the polymer's hydrophobic segments imposes entropy confinement on the crystallization behavior of the probe molecules. This causes the probe molecules to form closely packed H-aggregates within the micelle core, resulting in a decrease in fluorescence quantum yield to below 1% due to nonradiative transitions induced by molecular orbital coupling. This eliminates background fluorescence interference when traversing non-target tissues. Simultaneously, the random intercalation of the polycaprolactone segments blocks the path for probe molecules to adjust their orientation and form a long-range ordered lattice, maintaining the thermodynamically metastable amorphous state of the aggregates. When the concentration of the composition exceeds [a certain value], [further details are needed]. In a hydrogen peroxide environment, the thioketal bond breaks, removing the hydrophilic shell. The metastable probe molecule undergoes phase separation and dissociation driven by entropy increase, without needing to overcome the lattice energy barrier. The released probe monomer then inserts into the lattice. - The hydrophobic grooves of amyloid protein restrict intramolecular rotation and restore radiative transition channels, which increases the fluorescence emission intensity of the lesion area by more than 100 times relative to the background environment, thus achieving high-contrast imaging.

[0024] Example 2: In this example, an in vitro testing platform simulating a physiological and pathological microenvironment was constructed to quantitatively verify the optical response behavior of the composition of the present invention under different working conditions. The control test platform based on orthogonal design was designed to simulate high protein background interference in the vitreous humor and transient oxidative stress signals in the lesion area. The base medium simulating vitreous fluid consisted of a sodium hyaluronate solution with a concentration of 2.5 mg / mL, with the addition of human serum albumin with a concentration of 45.0 mg / mL to introduce a competitive interference source with strong hydrophobic binding ability. The data acquisition system used a high-sensitivity fluorescence spectrometer equipped with a temperature control unit, with the excitation wavelength locked at 480 nm and the emission spectrum scanning range set to 500 nm to 800 nm. To capture the millisecond-level micelle disassembly and assembly kinetics, the time resolution of the instrument was set to 100 ms.

[0025] To clarify the decisive impact of core technical features on performance, a test system was constructed that included the sample group of this invention and a multi-dimensional control group. The sample group of this invention (numbered S-10) followed the preferred technical scheme and used the number-average molecular weight of polycaprolactone segments. The probes were 2000 Da block copolymers with a probe loading mass ratio set at 10.0%. The control group C-Low (C-04) had its loading reduced to 4.0% to verify the effect of low loading on fluorescence quenching efficiency; the control group C-High (C-16) had its loading increased to 16.0% to investigate the hysteresis effect of supersaturated loading on release kinetics; and the control group C-Poly (C-4k) had its polycaprolactone segment molecular weight increased to 4000 Da to verify the inhibitory effect of the long-chain hydrophobic core on the response rate. The first stage of the experiment focused on assessing background fluorescence leakage during simulated transport. The samples were injected into simulated vitreous fluid at a constant temperature of 37°C and continuously stirred and monitored in the absence of an oxidizing trigger. The test data showed that the fluorescence quantum yield of the control group C-Low drifted to above 5.2% within the first 10 minutes and showed an increasing trend over time. This phenomenon indicates that when the loading is below the lower limit specified in this invention, the sparsely distributed probe molecules cannot form dense H-aggregates, making them susceptible to competitive capture by high concentrations of albumin, thus generating non-specific background signals. In contrast, the fluorescence quantum yields of the sample group S-10 and the high-load control group C-High of this invention were both stably maintained below 0.8%.

[0026] The second phase of the experiment examined the sensitivity of the compositions to pathological signals by instantaneously adding a final concentration of [missing information] to each of the above systems. Hydrogen peroxide solution, and introduce excess - Using amyloid fibrous protein as the probe receiving target, kinetic monitoring results revealed performance differences among the sample groups: Sample group S-10 of this invention showed an increase in fluorescence intensity within 2.5 seconds after triggering, rapidly reaching 124 times the initial background value, and reaching 90% of the peak intensity within 5 seconds. This data characteristic is consistent with the low energy barrier characteristic of frustrated H-aggregates transitioning from metastable to thermodynamically stable states. In contrast, although the control group C-High performed well during the quiescent period, its response after triggering was extremely slow, taking more than 18.5 minutes to reach 90% of the peak intensity. This time lag confirms that when the loading exceeds the upper limit, the core probe molecule transforms into a thermodynamically stable microcrystalline structure, and its dissociation process is constrained by a high lattice energy barrier. Similarly, the control group C-Poly's response time was extended to 8.2 minutes due to the excessively long hydrophobic segments enhancing the compactness of the micelle core. Table 1 summarizes the key performance indicators of each sample group.

[0027] Table 1: Comparison of Optical Response Performance of Different Groups

[0028] The above experimental data constructs a complete chain of evidence, objectively confirming that when the molecular weight of the polycaprolactone segment is limited to 1800 Da to 2200 Da and the probe loading is in a specific range of 8% to 12%, the composition can achieve both low background interference and fast response speed, two mutually restrictive key performance indicators.

[0029] Example 3: This example combines Figures 1 to 3 This describes a neurospecific combined detection composition for the assessment of cognitive impairment, such as... Figure 1 As shown, the key parameter for establishing physical confinement is the number-average molecular weight of the polycaprolactone segments. Limited to a value of 1800 Da to 2200 Da, a system consisting of a dispersed phase and a continuous phase was constructed based on this. The dispersed phase is a hydrophobic fluorescent probe with a mass ratio limited to 8:100 to 12:100, and the continuous phase is a reactive oxygen species (ROS) responsive block copolymer, namely methoxy polyethylene glycol-thioketal-polycaprolactone (mPEG-TK-PCL). The two phases self-assemble in an aqueous medium to form micelles with a hydrophobic core. This core forms a semi-crystalline microregion through physical confinement and encapsulates the probe, causing the probe to form thermodynamically metastable H-aggregates without sharp diffraction peaks, with a blue shift in UV absorption of 30 nm to 50 nm, and in a physically confined state. When the system is stimulated by high concentrations of reactive oxygen species (ROS) in a pathological environment, the thioketal bonds undergo chemical breakage, leading to the disassembly of the micelle structure, which in turn releases the probe and binds to the target amyloid protein, ultimately restoring a strong fluorescence signal.

[0030] like Figure 2 As shown, the fluorescence quenching efficiency (EQ) of different groups is compared on the left vertical axis (in percentage) with the signal enhancement factor. / A comparative analysis was performed on the right vertical axis. Sample group S-10, the preferred group of this invention, has an EQ value close to 100% and a signal enhancement factor more than 120 times higher than other control groups, indicating it is within the preferred operating window. In contrast, the low-load C-04 group has a lower EQ value and a signal enhancement factor of less than 20. While the high-load C-16 group has a higher EQ value, its signal enhancement factor is significantly lower than that of group S-10. Although the C-4K group using a long chain segment can also achieve high quenching, its signal enhancement factor is less than that of group S-10. Figure 3 As shown in the diagram, the microscopic mechanism illustrates the structural evolution of the composition from the transport state to the release state. In the transport state optical silencing on the left, the micelles exhibit a core-shell structure, with a hydrophilic methoxy polyethylene glycol (mPEG) segment on the outer layer and a hydrophobic polycaprolactone (PCL) segment on the inner layer. The two are connected by thioketal bonds, which serve as ROS-responsive cleavage sites. The hydrophobic core boundary encapsulates a core-shell structure. H-aggregate fluorescent probes that are stacked and in a fluorescence-quenched state will react with reactive oxygen species when they encounter reactive oxygen species. / When the response is triggered, the thioketal bond breaks, causing the micelles to disassemble and enter the released state fluorescence recovery on the right. At this time, the free PEG chain segments detach, and the released probe dissociates from the aggregate and binds to the hydrophobic microenvironment of the β-amyloid plaque. Affected by the folding structure of β-amyloid, the probe recovers fluorescence emission and is marked with an asterisk, thereby achieving specific labeling of the lesion.

[0031] Example 4: This example aims to provide a method for determining the number-average molecular weight of polycaprolactone segments. A standardized engineering calibration procedure with an optimized range was developed to eliminate potential uncertainties in the influence of this key structural parameter on microscopic phase control and fluorescence response performance. When developing micellar supports with frustrated H-aggregate cores, the length of the polycaprolactone chain segment determines the spatial confinement ability and crystallization inhibition efficiency of the hydrophobic microregions for probe molecules. An optimal working window exists that requires quantitative verification. The calibration process employs a gradient experimental design, controlling the molar ratio of monomer to initiator in the ring-opening polymerization reaction to synthesize a set of number-average molecular weight... The copolymers exhibit a gradient distribution of methoxylated polyethylene glycol-thioketal-polycaprolactone block copolymers. In each copolymer group, the molecular weight of the hydrophilic methoxylated polyethylene glycol is fixed at 2000 Da, while the molecular weight of the hydrophobic polycaprolactone segments... The hydrophobic fluorescent probes were set to 1500 Da, 1800 Da, 2000 Da, 2200 Da and 2500 Da respectively. Following the restricted antisolvent kinetics capture process in Example 1, hydrophobic fluorescent probes with a fixed loading of 10.0% were loaded into the copolymer micelles of the above groups to prepare the corresponding test sample groups.

[0032] Dual performance tests were conducted on the background fluorescence during transport and the release kinetics during the response period for each sample group. The test data showed that when the polycaprolactone segment... At 1500 Da, the background fluorescence quantum yield of the composition is as high as 3.8%, exceeding the silencing threshold of 1%. The mechanism lies in the fact that the hydrophobic segments are too short to provide sufficient steric hindrance to induce the formation of dense H-aggregates of probe molecules, resulting in some probes existing in monomeric or loosely aggregated states. Increasing to 1800 Da and 2000 Da, the background fluorescence quantum yield rapidly decreased to below 0.8%, while at the contact... Within 2.5 seconds of the hydrogen peroxide trigger, the fluorescence intensity recovered to over 90% of its peak value, demonstrating excellent frustrated metastable characteristics. However, when... When the value was further increased to 2500 Da, although the background fluorescence remained at a low level, the response time... The response time was extended to over 320 seconds, indicating that the excessively long hydrophobic segments enhanced the compactness of the micelle core and the intermolecular entanglement, hindering the rapid dissociation of the micelles after the hydrophilic shell detached, resulting in a sluggish response.

[0033] Example 5: Before the composition of the present invention is mass-produced and put into actual medical application, in order to eliminate the potential impact of slight differences between batches of raw materials on the efficiency of frustrated H-aggregate formation, a standardized initial phase calibration procedure is performed. The core of this procedure is to compensate for the entropy limitation deviation introduced by the possible fluctuations in molecular weight distribution during polymer synthesis by fine-tuning the component mass ratio. For each batch of newly synthesized methoxy polyethylene glycol-thioketal-polycaprolactone copolymer, the actual number-average molecular weight of its polycaprolactone segments is determined by gel permeation chromatography. and the polydispersity index (PDI), if measured If the value deviates from the 2000 Da benchmark but remains within the allowable range of 1800 Da to 2200 Da, the loading mass ratio of the hydrophobic fluorescent probe needs to be adjusted according to a specific compensation logic: when If the value is too low, the probe loading should be appropriately increased within the range of 8% to 12% to enhance the packing density within the hydrophobic core and compensate for the insufficient steric hindrance; define the segment-probe interaction parameters under standard conditions. Compared with the reference free volume When the gel permeation chromatography value is measured Below the benchmark value At that time, the decrease in the spatial confinement capability of the hydrophobic micro-region The error is exponentially correlated with the molecular weight deviation, and the compensation logic executes the correction formula. ,in, Standard load Sensitivity coefficient After experimental calibration, it was determined to be If the calculated result If the polymer chain segment regularity of this batch is insufficient to maintain the metastable energy barrier of the frustrated H-aggregates, then the batching process will be terminated and the raw materials will be scrapped; otherwise... Perform reverse compensation ,in: This represents the actual mass ratio of the hydrophobic fluorescent probe loading after calibration. This is the standard load, with a value of 10.0%. The square correction factor is used to characterize the nonlinear contribution of increased hydrophobic segment density to the probe repulsion effect. Its value was determined experimentally. ; The actual number-average molecular weight of the polycaprolactone segments was measured. The baseline number-average molecular weight is set at 2000 Da. Adjusting the loading using this formula prevents excessively high chain density from causing probe repulsion, ensuring the probe intermolecular distance remains consistently anchored. The fluorescence quenching region, and conversely, when If the concentration is too high, the loading should be appropriately reduced to prevent excessive chain segment entanglement from hindering the dissociation and release of the probe. The adjusted formulation needs to be prepared on a small scale, and the maximum absorption wavelength should be detected by UV-Vis absorption spectroscopy. The degree of blue shift is used to confirm whether it meets the preset H-aggregate determination criteria. When the blue shift value is stable within the range of 30nm to 50nm, the batch of formulation can be confirmed as calibrated and can proceed to the subsequent production process.

[0034] In addition, to ensure the long-term stability of the composition under different storage and transportation conditions, a baseline monitoring procedure for the thermodynamic metastable state of frustrated H-aggregates needs to be established. This requires that after the finished product is prepared, samples be taken periodically for X-ray powder diffraction analysis to monitor for the presence of characteristic diffraction peaks corresponding to the probe crystal structure. If sharp diffraction peaks are detected within the specified shelf life, it indicates that the amorphous aggregates in the micelle core have undergone a phase transition to a thermodynamically stable microcrystalline state. At this time, an early warning mechanism is activated, and the solvent removal rate and temperature fluctuations in the storage environment are reviewed retrospectively. At the same time, the average hydrated particle size and potential distribution of the micelles are continuously monitored through dynamic light scattering technology, and a correlation database between particle size changes and phase stability is established. Once abnormal broadening or bimodal phenomena in the particle size distribution are found, it is determined to be a precursor to micelle structural instability, and the batch of products needs to be isolated and retested.

[0035] Example 6: In clinical applications where detection is adapted to the differences in fundus anatomy among different subjects, the inherent differences in retinal thickness and fundus media scattering coefficients between individuals present an objective challenge of signal-to-noise ratio imbalance when setting uniform excitation intensity and response threshold. If the compensation of individualized optical attenuation factors is ignored, subjects with thicker retinas or higher media turbidity may experience false negatives, while subjects with stronger choroidal reflection may experience false positives. To eliminate this detection error, this example provides a pre-deployment calibration and parameter calibration procedure based on optical coherence tomography. By introducing individualized correction factors to fine-tune the response baseline of the fluorescent probe, consistent detection sensitivity is ensured among different subjects.

[0036] The procedure begins with a baseline scan of the macular region of the subject's retina. A frequency-domain optical coherence tomography (OCT) system is used to acquire retinal tomographic images within a 1mm radius of the fovea, and the average thickness of the neuroepithelial layer is measured. and its scattering coefficient for a specific wavelength of light Based on a pre-established quantitative model, the combined attenuation factor of the subject's retinal environment on 480nm excitation light and 650nm emission light was calculated. According to the formula Calculate the probe concentration adjustment value suitable for this subject. In the formula, The standard neuroepithelial thickness is set as a baseline value, and a ratio is introduced. The probe dosage is linearly compensated for based on the target tissue volume, and the scattering coefficient is... As an individualized moderating variable, the proportionality correction coefficient k is used to map the subject-specific scattering contribution to the dose adjustment space; the attenuation factor... These are not empirical values; they must be derived from the integral form of the Beer-Lambert law in multilayer scattering media. The raw A-scan signal from optical coherence tomography is extracted, and the segment from the retinal nerve fiber layer to the retinal pigment epithelium layer is selected to calculate the depth-resolved backscattering intensity. Using the formula Solve for the local scattering coefficient Distribution; Construct a photon transport model and utilize integral paths Total thickness of retinal tissue Together, we define the physical boundaries of the detection field and define the comprehensive attenuation factor. ,in, The medium absorption coefficient, For the reduced scattering coefficient This is the overall attenuation factor; The total thickness of the retinal tissue is used as the upper limit of integration to determine the solution boundary for attenuation calculation; The independent variable is the integral along the depth direction of the retina; The excitation wavelength is set to 480 nm. The emission wavelength is set to 650nm. For wavelength In Depth The medium absorption coefficient at that location; For wavelength In Depth Reduced scattering coefficient at the point, integration path The entire optical path from the vitreous body to the outer nuclear layer of the retina is covered to define the spatial extent of absorption and scattering in the photon transport model; if the calculated... And local scattering peaks If the subject is determined to have cataracts or vitreous opacity, the logic judgment module automatically outputs a warning of insufficient signal-to-noise ratio and suggests intravitreal injection as an alternative to eye drops; when Then, the numerical values ​​are substituted into the concentration adjustment formula to complete the precise calculation of the formulation dosage based on individual optical characteristics. In the formula, The standard probe loading concentration, This is the proportional correction factor, if If the concentration exceeds the allowable range of the standard formulation, it is fine-tuned by on-site dilution or by adding a specific concentration of adjuvant enhancer. A small amount of the adjusted formulation is then used for rapid response testing in an in vitro simulated fundus solution to confirm the deviation between its fluorescence recovery kinetic curve and the standard curve. Only after this process is complete can medication be administered.

[0037] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A neurospecific combined detection composition for assessing cognitive impairment, characterized in that, The composition is an ophthalmic formulation comprising a dispersed phase and a continuous phase medium: The dispersed phase is a hydrophobic fluorescent probe, and the continuous phase medium is a reactive oxygen species-responsive amphiphilic block copolymer; The reactive oxygen species responsive amphiphilic block copolymer is methoxy polyethylene glycol-thioketal-polycaprolactone, which assembles in an aqueous medium to form micelles with a hydrophobic core, and the hydrophobic fluorescent probe is encapsulated in the hydrophobic core. Among them, the number average molecular weight of the polycaprolactone segment in methoxy polyethylene glycol-thioketone-polycaprolactone is limited to 1800 Da to 2200 Da, so as to form a semi-crystalline micro-region that physically confines the hydrophobic fluorescent probe in the hydrophobic core. The mass ratio of the hydrophobic fluorescent probe to the reactive oxygen species-responsive amphiphilic block copolymer is limited to 8:100 to 12:100; Based on the physical confinement effect of mass ratio and polycaprolactone segments, the hydrophobic fluorescent probe forms thermodynamically metastable H-aggregates in the hydrophobic core. The thermodynamically metastable H-aggregates do not have sharp diffraction peaks corresponding to the single crystal of the hydrophobic fluorescent probe in the X-ray powder diffraction pattern, but show diffuse peaks of the polymer matrix. Moreover, the maximum absorption wavelength in the UV-Vis absorption spectrum is blue-shifted by 30 nm to 50 nm relative to the monomer solution of the hydrophobic fluorescent probe. When the thioketal bond breaks in response to pathological concentrations of reactive oxygen species, the hydrophobic fluorescent probe is released along with the micelle disintegration and binds to the target amyloid protein.

2. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The hydrophobic fluorescent probe is selected from one of the modified curcumin derivatives or boron fluoride dipyrrole derivatives with molecular rotor characteristics, whose molecular structure contains at least two rigid conjugated planes capable of forming π-π stacking; the number average molecular weight of the methoxy polyethylene glycol segment in methoxy polyethylene glycol-thioketal-polycaprolactone is 1900 Da to 2100 Da; the fluorescence quantum yield of the composition in the thermodynamically metastable H-aggregate state is less than 1%, and the fluorescence recovery response time after the thioketal bond breaks is less than 100 ms; the particle size distribution coefficient of the composition is less than 0.

2.

3. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The composition was prepared by an antisolvent precipitation process, which included: dissolving a hydrophobic fluorescent probe and a reactive oxygen species-responsive amphiphilic block copolymer in tetrahydrofuran to form a homogeneous solution; injecting the homogeneous solution into deionized water at a flow rate of 10.0 mL / min to 12.0 mL / min while stirring at a speed of 1000 rpm to 1200 rpm; and fixing the hydrophobic fluorescent probe in a thermodynamically metastable H-aggregate state by matching the flow rate with the precipitation rate of the polycaprolactone segments.

4. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The background fluorescence suppression capability of the composition is determined by the fluorescence quenching efficiency. Characterization, fluorescence quenching efficiency The following relationship must be satisfied: ,in, The fluorescence emission intensity of the composition at the excitation wavelength is measured when the composition is in an intact micelle state. The fluorescence emission intensity is the value of the composition after the thioketal bond is completely broken and the hydrophobic fluorescent probe is completely released into the octanol medium.

5. The neurospecific combined detection composition for cognitive impairment assessment according to claim 3, characterized in that, The volume ratio of tetrahydrofuran to deionized water is 1:5 to 1:10; the process also includes open stirring of the mixed solution at an ambient temperature of 30 degrees Celsius to remove tetrahydrofuran, and dialysis purification of the micelle solution using a dialysis unit with a molecular weight cutoff of 3500 Da. Thermodynamically metastable H-aggregates are amorphous structures formed based on solvent displacement and competition for crystallization of polycaprolactone segments.

6. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The composition also includes a pharmaceutically acceptable isotonic buffer solution with a pH of 7.0 to 7.4; the composition exhibits a fluorescence leakage rate of less than 2% within 30 days when stored at 4 degrees Celsius in the dark. The hydrophobic fluorescent probe reacts with... When amyloid plaques bind, their fluorescence emission intensity increases by at least 100 times relative to the thermodynamically metastable H-aggregate state before release, achieving a dual specific response to oxidative stress and amyloid deposition.

7. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The critical micelle concentration of reactive oxygen species-responsive amphiphilic block copolymers is mg / mL to mg / mL; the thioketal bond is located at the junction of the methoxy polyethylene glycol segment and the polycaprolactone segment, and is used to chemically break in an environment where the concentration of hydrogen peroxide or hydroxyl radicals exceeds 100 μmol / L, thereby leading to micelle disassembly.

8. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The emission wavelength of the hydrophobic fluorescent probe is in the region of 650 nm to 800 nm, and its Stokes shift is greater than 80 nm. The intermolecular distance of the hydrophobic fluorescent probe in the thermodynamically metastable H-aggregate state is restricted by polymer chain segments to between 0.3 nm and 0.4 nm. This distance is less than the lattice spacing required to form a stable crystal but is sufficient to induce intermolecular interactions to achieve fluorescence quenching.

9. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The composition is used to prepare a fundus diagnostic reagent for assessing the risk of Alzheimer's disease or mild cognitive impairment. The reagent is delivered to the retina via eye drops to avoid nonspecific fluorescence interference from the lens and vitreous body.

10. The neurospecific combined detection composition for cognitive impairment assessment according to claim 1, characterized in that, The composition is a colloidal solution with a zeta potential of -10 mV to -20 mV; the residual organic solvent content in the composition is less than 50 parts per million; the thermodynamically metastable H-aggregates still maintain the amorphous characteristics without sharp crystal diffraction peaks in the X-ray powder diffraction pattern after three freeze-thaw cycles, indicating that the polycaprolactone chain segment has reversible and stable inhibitory effect on the crystallization of hydrophobic fluorescent probes.

Images